A kind of pcr detection method of bumble bee sporozoite
A technology of sporozoites and bumblebees, which is applied in the field of PCR detection of bumblebee sporozoites, can solve the problems of confusion and easy missed detection, and achieve the effect of strong specificity and good stability
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Embodiment 1
[0022] Example 1 The specificity test of Bombus sporozoites
[0023] By adopting the method of the invention, the bumblebee sporozoite is tested for the DNA samples and other parasitic diseases and pests whose amplified sequence is positive for the bumblebee sporozoite.
[0024] Specific primer pairs are:
[0025] Api-F: 5'-AGCGATGGATGTCTTGGG-3';
[0026] Api-R: 5'-TTCAGCGGGTAACCTTGT-3'.
[0027] The PCR reaction system included 4 μL of 5×GoTaq Buffer, 1.5 μL of MgCl 2 (25mM) (Promega), 2.0 μL of dNTPs (200 μM) (TaKaRa), 1 μL each of primer Api-F and primer Api-R (10 μM), 0.5 μL of GoTaq® DNA polymerase, 2 μL of template DNA , ddH 2 O to make up to 20 μL:
[0028] The reaction conditions were pre-denaturation at 95°C for 4 min, followed by 40 cycles of denaturation at 95°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 10 min.
[0029] Take 6 μL of the amplification product and detect it by 1.5% agarose gel electro...
Embodiment 2
[0030] Example 2 Sensitivity test of Bombus sporozoites
[0031] By adopting the method of the present invention, the sensitivity test is carried out on different dilution multiples of the DNA of the whole gene synthesis of the bumblebee sporozoite.
[0032] The PCR reaction method, system and conditions are the same as in Example 1, and the PCR amplification product is detected by 1.5% agarose gel electrophoresis. figure 2 Bombus sporozoite DNA10 synthesized for whole gene 0 -10 15 Agarose gel electrophoresis bands of PCR products after doubling dilution. The PCR product was detected by 1.5% agarose gel electrophoresis and found that 10 8 Bands were amplified from the 139 ng / μL template DNA diluted twice, and the sensitivity of the specific primers in the present invention to Api-F / Api-R was 1.39 fg / μL.
Embodiment 3
[0033] Example 3 Detection of clinical samples of Bombus sporozoites
[0034] Extract the genomic DNA from the abdominal tissue of the bumblebee to be tested
[0035] Cut the bumblebee abdominal tissue and place it in a mortar, add an appropriate amount of liquid nitrogen to quickly grind it, grind it several times to form a slurry, and use the CTAB method to extract the bumblebee abdominal tissue genomic DNA. The specific steps are as follows:
[0036] The bumblebee abdominal tissue was ground into a slurry and transferred to a 1.5 mL centrifuge tube, centrifuged at 12000 rpm for 2 min, the supernatant was discarded, and 50 µL of TE buffer was added to fully suspend the sample, then 60 µL of 10% SDS solution and 10 µL of 20 mg / mL proteinase K, mix well and incubate at 37°C for 1 h;
[0037] Add 100 µL of 5 mol / L NaCl solution, mix well by inverting up and down, add 80 µL CTAB / NaCl solution, and incubate at 65°C for 10 min after mixing;
[0038] Add 700 µL chloroform / isoamy...
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